Polypeptide and use thereof

ABSTRACT

Provided are a polypeptide and nucleic acid for encoding the polypeptide, a nucleic-acid construct, an expression vector, and a host cell containing the nucleic acid, an antigen-presenting cell presenting the polypeptide on the surface of the cell, and immune effector cell thereof, a pharmaceutical composition containing the polypeptide, a vaccine containing the nucleic acid, the nucleic acid construct, the expression vector, the host cell, the antigen-presenting cell, and the immune effector cell, and an antibody recognizing the polypeptide. Also provided is a therapeutic method using the polypeptide, the nucleic acid, the pharmaceutical composition, the vaccine, and the antibody. Also provided are a diagnosis method and diagnosis apparatus for detecting the described polypeptide. Also provided is an application of the polypeptide in preparing a vaccine, a tumor diagnosis kit, or a pharmaceutical composition, and an application of the polypeptide or the nucleic acid as a test target in tumor diagnosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/417,579, filed May 20, 2019, which is a continuation of InternationalApplication No. PCT/CN2016/106797, filed Nov. 22, 2016, the entiredisclosures of which are incorporated herein by reference.

FIELD

The present disclosure relates to the field of biomedicine, inparticular to a polypeptide and use thereof; more particular to apolypeptide and use thereof in the preparation of a kit, a medicamentand a vaccine, and to the use thereof in preventing or treating adisease associated with a mutation of GLRA2 gene in a subject; to anucleic acid, a nucleic acid construct, an expression vector, a hostcell, a pharmaceutical composition, an antigen-presenting cell, animmune effector cell, a vaccine, an antibody; and to a therapeuticmethod, a diagnostic method and a diagnostic system.

BACKGROUND

Cancer is a disease involving abnormal cell proliferation due to geneticmutation in cells, which has become one of major threats and a maincause to human health. In the Global Cancer Report 2014 distributed bythe World Health Organization (WHO), it is indicated that the number ofglobal cancer patients and cancer deaths are in a rapid increase in2012, and nearly half of the newly-diagnosed patients are Asianpopulations, especially from China (which ranks the first innewly-diagnosed cancer cases). It is also showed by data in the ChinaCancer Registry Annual Report 2012 that the number of annualnewly-diagnosed cancer patients is about 3.5 million, and 2.5 millionpatients of which even progress to death. Thus, it has an importantclinical value to discover an effective and specific cancer therapy.

Traditional tumor therapies mainly include surgery, radiotherapy andchemotherapy, however all of them have great limitations. For example, ahigh tumor metastasis recurrence rate after surgical resection is mainlydue to proximal invasion or distant metastasis of cancer cells, andradiotherapy and chemotherapy will cause severe damage to normal cellsin the body, especially to the hematopoietic system and immune system,thus it is difficult to achieve a better long-term curative effect forpatients with tumor metastasis. With an in-depth study of the molecularmechanism of tumor and the development of biotechnology, targetedtherapy (which mainly involves monoclonal antibody classified as apassive immunotherapy sometimes, and small molecule targeted drugs) andimmunotherapy (which mainly includes cytokine therapy, immune checkpointinhibitor, adoptive cell transfer, tumor vaccine and the like) areincreasingly playing an important role in the integrated tumortreatment, in which immunotherapy enhances anti-tumor immunity in atumor microenvironment by regulating the immune system of the body,thereby inhibiting and killing tumor cells in a highly effective andspecific way with good tolerance, thus it has a wide prospect in tumortherapy.

Tumor immunotherapeutic vaccines mainly include tumor cell vaccine,dendritic cell (DC cell) vaccine, protein & polypeptide vaccine, nucleicacid vaccine, and genetic engineering vaccine, which will kill tumorsmainly by mechanisms of inducing an immune response by thetumor-specific as antigen in a patient, including an antigen-antibodyreaction and specific killing of cytotoxic T lymphocyte (CTL) whichplays a vital role in the tumor immune response. A tumor-specificpolypeptide, as a tumor-specific antigen which will induce specific CTLkilling, includes tumor-mutant polypeptides and tumor-specificoverexpressed polypeptides. The tumor-mutant polypeptide, which is onlypresented in tumor of a patient, is a specific target of tumorimmunotherapy, with good safety and low side effect. The immunotherapytargeting the tumor-mutant polypeptide is represented bypolypeptide-specific DC-CTL and adoptive transfer of tumor infiltratinglymphocytes (TIL), and exhibits a good therapeutic effect.

The tumor-specific polypeptides can be recognized by CTL cells or TILcells under the presentation of human leukocyte antigen (HLA). The HLAsare divided into two classes (HLA-I and HLA-II), where the HLA-I classincludes three subtypes HLA-A, HLA-B and HLA-C and each of them can alsohave several subtypes depending on different sequences, for example, oneof subtype HLA-As is HLA-A0201, which is in a high percent (accountingfor 13%) in Chinese populations. Different polypeptides have differentbinding activity to HLA-A0201. In tumor patients having specific HLAsubtypes, only a portion of mutant polypeptides can be bond tocorresponding HLAs and followed by presented to CTL cells or TIL cellsvia HLAs depending on the HLA subtypes.

However, there still remains a need to further research and develop thetumor immunotherapy.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of theproblems existing in the related art to at least some extent.

In one aspect of the present disclosure, provided in embodiments is anisolated polypeptide of SEQ ID NO: 1 (TLAFLIFNI) or a fragment thereof,where the fragment includes at least 9 amino acids which has at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% or at least 99% sequence identity to the isolated polypeptide of SEQID NO: 1, or has one or more of amino acid substitution(s), deletion(s)and addition(s) compared to the isolated polypeptide of SEQ ID NO: 1.

In one embodiment of the present disclosure, the one or more of aminoacid substitution(s), deletion(s) and/or addition(s) is at least one ofamino acid substitution at position 2 and amino acid substitution atposition 9 in the amino acid sequence as depicted in SEQ ID NO: 1.

In one embodiment of the present disclosure, the one or more of aminoacid substitution(s), deletion(s) and/or addition(s) is at least one ofsubstitution with Methionine at position 2 and substitution with Valineor Leucine at position 9 in the amino acid sequence as depicted in SEQID NO: 1.

In one embodiment of the present disclosure, the fragment has 9 aminoacids as depicted in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

(SEQ ID NO: 1) TLAFLIFNI. (SEQ ID NO: 2) TMAFLIFNI. (SEQ ID NO: 3)TLAFLIFNV. (SEQ ID NO: 4) TLAFLIFNL. (SEQ ID NO: 5) TMAFLIFNL.(SEQ ID NO: 6) TMAFLIFNV.

In embodiments of the present disclosure, the polypeptide binds withhigh affinity to HLA-A0201, thus having the ability to activate specificT cell immunity.

In another aspect of the present disclosure, provided in embodiments isuse of a reagent for detecting the polypeptide as described above in thepreparation of a kit for diagnosing a tumor. Optionally, the polypeptideis expressed in a tumor from a HLA-A0201 positive subject. Optionally,the tumor is one or more selected from the group consisting of breastcancer, lung cancer, nasopharyngeal carcinoma, liver cancer, gastriccancer, esophageal cancer, colorectal cancer, pancreatic cancer,melanoma, skin cancer, prostate cancer, cervical cancer, leukemia andbrain tumor. Preferably, the tumor is non-small cell lung cancer. Basedon the experiments and investigations, it is found by the presentinventors that the polypeptide as described above is specificallyoverexpressed in a tumors. Further, it is verified through experimentsand proposed by the present inventors that the kit prepared with thereagent for detecting the polypeptide as described above can be usefulin diagnosing a tumor effectively. Meanwhile, it is surprisinglydiscovered by the present inventors that the polypeptide binds with highaffinity to HLA-A0201 and thus can be presented by HLA-A0201-expressingpresenting cells to CTL cells or TIL cells for activation of specific Tcell immunity, therefore when the polypeptide is expressed in a tumorfrom a HLA-A0201 positive subject, the safety and effectiveness ofdiagnosis by the kit can be significantly improved. Further, it is alsodiscovered by the present inventors that the polypeptide is specificallyoverexpressed in a tumor selected from one or more of breast cancer,lung cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer,esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skincancer, prostate cancer, cervical cancer, leukemia and brain tumor, thuswhen the tumor as described above is diagnosed, the effectiveness andsensitivity of diagnosis by the kit can be further improved.

In another aspect of the present disclosure, provided in embodiments isuse of the polypeptide as described above in the preparation of amedicament for preventing or treating a tumor. Optionally, thepolypeptide is expressed in a tumor from a HLA-A0201 positive subject.Optionally, the tumor is one or more selected from the group consistingof breast cancer, lung cancer, nasopharyngeal carcinoma, liver cancer,gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer,melanoma, skin cancer, prostate cancer, cervical cancer, leukemia andbrain tumor. Preferably, the tumor is non-small cell lung cancer. Asdescribed above, it is found by the present inventors that thepolypeptide as described above is specifically overexpressed in a tumor.Further, it is verified through experiments and proposed by the presentinventors that the medicament prepared with the polypeptide as describedabove can be useful in effectively preventing or treating a tumor. It isfound that when the polypeptide is expressed in a tumor from a HLA-A0201positive subject, the safety and effectiveness of the treatment orprevention by the medicament are significantly improved. Furthermore, ifthe tumor is one or more selected from the group consisting of breastcancer, lung cancer, nasopharyngeal carcinoma, liver cancer, gastriccancer, esophageal cancer, colorectal cancer, pancreatic cancer,melanoma, skin cancer, prostate cancer, cervical cancer, leukemia andbrain tumor, especially non-small cell lung cancer, the effectivenessand sensitivity of the treatment or prevention by the medicament can befurther improved.

In another aspect of the present disclosure, provided in embodiments isan isolated nucleic acid.

According to an embodiment of the present disclosure, the isolatednucleic acid is a nucleic acid encoding the polypeptide or the fragmentthereof as described above or a complement thereof, where the isolatednucleic acid is capable of specifically encoding the polypeptide. Asdescribed above, the polypeptide binds with high affinity to HLA-A0201and thus can be presented by HLA-A0201-expressing presenting cells toCTL cells or TIL cells for activation of specific T cell immunity.Therefore, the polypeptide expressed by the isolated nucleic acidproposed in embodiments of the present disclosure under a suitablecondition can be used to prevent or treat a tumor, especially thepolypeptide-expressing tumor from the HLA-A0201 positive subject, thesafety and effectiveness of the treatment or prevention are improved.

In another aspect of the present disclosure, provided in embodiments isa nucleic acid construct.

According to an embodiment of the present disclosure, the nucleic acidconstruct includes an encoding sequence, wherein the encoding sequenceis the nucleic acid as described above, and optionally a controlsequence operably connected to the encoding sequence, wherein thecontrol sequence is one or more control sequence(s) capable of directingthe expression of the polypeptide in a host cell. Therefore, aftertransfected or infected under a suitable condition with the nucleic acidconstruct proposed in embodiments of the present disclosure which isconnected with a vector, the suitable host cell can express thepolypeptide as described above, thus being capable of specificallypreventing or treating a tumor in an effective way, particularly thepolypeptide-expressing tumor from the HLA-A0201 positive subject.

In another aspect of the present disclosure, provided in embodiments isan expression vector.

According to an embodiment of the present disclosure, the expressionvector includes the nucleic acid construct as described above.Therefore, after transfected or infected with the expression vectorproposed in embodiments of the present disclosure under a suitablecondition, the host cell can efficiently express the polypeptide asdescribed above, thus being capable of specifically preventing ortreating a tumor in an effective way, particularly thepolypeptide-expressing tumor from the HLA-A0201 positive subject.

In another aspect of the present disclosure, provided in embodiments isa host cell. According to an embodiment of the present disclosure, thehost cell carries the nucleic acid construct or the expression vector asdescribed above, optionally obtained by transfecting or transforming thenucleic acid construct or the expression vector. According to anembodiment of the present disclosure, the host cell can efficientlyexpress the polypeptide as described above under a suitable condition,thus being capable of specifically preventing or treating a tumor in aneffective way, particularly the polypeptide-expressing tumor from theHLA-A0201 positive subject.

In another aspect of the present disclosure, provided in embodiments isa pharmaceutical composition.

According to an embodiment of the present disclosure, the pharmaceuticalcomposition includes the polypeptide as described above, and apharmaceutically acceptable adjuvant. It is discovered by the presentinventors through a lot of experiments that the pharmaceuticalcomposition including the polypeptide as described above and thepharmaceutically acceptable adjuvant can significantly stimulate theproliferation and secretion of CTL cells or TIL cells, remarkablykilling the tumor cell which expresses the polypeptide as an antigen,thus exhibiting an efficacy of significantly preventing or treating atumor, particularly the tumor specifically overexpressing thepolypeptide as an antigen.

In another aspect of the present disclosure, provided in embodiments isuse of the polypeptide as described above in the preparation of avaccine for preventing or treating a tumor. Optionally, the polypeptideis expressed in a tumor from a HLA-A0201 positive subject. Optionally,the tumor is one or more selected from the group consisting of breastcancer, lung cancer, nasopharyngeal carcinoma, liver cancer, gastriccancer, esophageal cancer, colorectal cancer, pancreatic cancer,melanoma, skin cancer, prostate cancer, cervical cancer, leukemia andbrain tumor. Preferably, the tumor is non-small cell lung cancer. Asdescribed above, it is found by the present inventors that thepolypeptide as described above is specifically overexpressed in a tumor.Further, it is verified through experiments and proposed by the presentinventors that the vaccine prepared with the polypeptide as describedabove can be useful in effectively preventing or treating a tumor, withhigher safety and lower side effect. It is found that when thepolypeptide is expressed in a tumor from a HLA-A0201 positive subject,the safety and effectiveness of the treatment or prevention by thevaccine are significantly improved. Furthermore, if the tumor is one ormore selected from the group consisting of breast cancer, lung cancer,nasopharyngeal carcinoma, liver cancer, gastric cancer, esophagealcancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer,prostate cancer, cervical cancer, leukemia and brain tumor, especiallynon-small cell lung cancer, the effectiveness and sensitivity of thetreatment or prevention by the vaccine can be further improved.

In another aspect of the present disclosure, provided in embodiments isan antigen-presenting cell.

In an embodiment of the present disclosure, the antigen-presenting cellpresents the polypeptide as described above. According to an embodimentof the present disclosure, the antigen-presenting cell which presentsthe polypeptide described above can effectively induce an immuneresponse by the tumor-specific polypeptide as described above (asantigen) in a subject, thus activating the function of specific CTLkilling. The antigen-presenting cell proposed in embodiments of thepresent disclosure has a significant efficacy of treating thepolypeptide-expressing tumor from the HLA-A0201 positive subject withhigher safety.

In another aspect of the present disclosure, provided in embodiments isan immune effector cell.

According to an embodiment of the present disclosure, the immuneeffector cell can recognize the polypeptide as described above or theantigen-presenting cell presenting the polypeptide as described above onits surface. According to an embodiment of the present disclosure, theimmune effector cell specifically kills the polypeptide-expressing tumorfrom the HLA-A0201 positive subject.

In another aspect of the present disclosure, provided in embodiments isa vaccine.

According to an embodiment of the present disclosure, the vaccineincludes the nucleic acid, the nucleic acid construct, the expressionvector, the host cell, the antigen-presenting cell, or the immuneeffector cell as described above. As mentioned above, after transfectedor infected with the nucleic acid, the nucleic acid construct or theexpression vector proposed in embodiments of the present disclosureunder a suitable condition, the host cell can express the polypeptide asdescribed above, thus the nucleic acid, the nucleic acid construct andthe expression vector in embodiments of the present disclosure each canbe used for preventing or treating the polypeptide-expressing tumor;further the antigen-presenting cell proposed in embodiments of thepresent disclosure has a significant efficacy in treating thepolypeptide-expressing tumor from the HLA-A0201 positive subject; andfurthermore the immune effector cell proposed in embodiments of thepresent disclosure has a significant efficacy in specifically killing atarget cell expressing the polypeptide as an antigen. The vaccineproposed in embodiments of the present disclosure has a significantefficacy of preventing or treating the polypeptide-expressing tumor fromthe HLA-A0201 positive subject with higher safety and lower side effect.

In another aspect of the present disclosure, provided in embodiments isan antibody.

According to an embodiment of the present disclosure, the antibodyspecifically recognizes the polypeptide as described above. The antibodyproposed in embodiments of the present disclosure can specifically bindto said polypeptide, thereby being capable of specifically identifying atumor cell which specifically overexpresses the polypeptide, thus theantibody proposed in the embodiment of the present disclosure plays ahuge role in tumor diagnosis, treatment or prevention.

In another aspect of the present disclosure, provided in embodiments isa therapeutic method.

According to embodiments of the present disclosure, the therapeuticmethod includes administering to a subject a therapeutically effectiveamount of the polypeptide, the nucleic acid, the nucleic acid construct,the expression vector, the host cell, the pharmaceutical composition,the antigen-presenting cell, the immune effector cell, the vaccine orthe antibody as described above. As mentioned above, the therapeuticmethod proposed in embodiments of the present disclosure includingadministering a therapeutically effective amount of any of thepolypeptide, the nucleic acid, the nucleic acid construct, theexpression vector, the host cell, the pharmaceutical composition, theantigen-presenting cell, the immune effector cell, the vaccine and theantibody as described above is capable of effectively treating orpreventing the polypeptide-expressing tumor from the HLA-A0201 positivesubject.

In another aspect of the present disclosure, provided in embodiments isuse of the polypeptide as described above in the prevention or treatmentof a disease associated with a mutation of GLRA2 gene in a subject. Thepolypeptide in embodiments of the present disclosure useful inpreventing or treating a disease associated with a mutation of GLRA2gene in a subject exhibits a significant efficacy.

In another aspect of the present disclosure, provided in embodiments isa diagnostic method.

According to an embodiment of the present disclosure, the diagnosticmethod includes: detecting whether a biological sample derived from asubject contains the polypeptide or the fragment thereof as describedabove, and determining whether the subject suffers from a tumor based onthe presence or absence of the polypeptide in the biological sample,optionally the polypeptide is expressed in a tumor from a HLA-A0201positive subject, and optionally the tumor is one or more selected fromthe group consisting of breast cancer, lung cancer, nasopharyngealcarcinoma, liver cancer, gastric cancer, esophageal cancer, colorectalcancer, pancreatic cancer, melanoma, skin cancer, prostate cancer,cervical cancer, leukemia and brain tumor. Preferably, the tumor isnon-small cell lung cancer. It is found by the present inventors thatthe polypeptide is specifically overexpressed in a tumor, whereas suchthe polypeptide is absent in a tumor-free tissue, thus a subject with atumor specifically overexpressing the polypeptide can be effectivelydiagnosed by the diagnostic method proposed in embodiments of thepresent disclosure. It is further discovered by the present inventorsthat the polypeptide is specifically overexpressed in a tumor selectedfrom one or more of breast cancer, lung cancer, nasopharyngealcarcinoma, liver cancer, gastric cancer, esophageal cancer, colorectalcancer, pancreatic cancer, melanoma, skin cancer, prostate cancer,cervical cancer, leukemia and brain tumor, thus the accuracy ofdiagnosing the tumor as described above can be further improved by usingthe diagnostic method proposed in the embodiment of the presentdisclosure. Meanwhile, it is demonstrated by the present inventors thatHLA-A0201 is present in Chinese populations in a high proportion, whichbinds with strong affinity to the polypeptide for activation of aserious of immune responses, thus the accuracy of diagnosis of thepolypeptide-expressing tumor from the HLA-A0201 positive subject isimproved by using the diagnostic method proposed in the embodiment ofthe present disclosure.

In another aspect of the present disclosure, provided in embodiments isa diagnostic system.

According to an embodiment of the present disclosure, the diagnosticsystem includes a polypeptide detecting device, configured to detectwhether a biological sample derived from a subject contains thepolypeptide as described above; and a diagnosis determining device,connected to the polypeptide determining device and configured todetermine whether the subject suffers from a tumor based on the presenceor absence of the polypeptide in the biological sample.

In an embodiment of the present disclosure, the polypeptide is expressedin a tumor from a HLA-A0201 positive subject.

In an embodiment of the present disclosure, the tumor described in thisaspect is one or more selected from the group consisting of breastcancer, lung cancer, nasopharyngeal carcinoma, liver cancer, gastriccancer, esophageal cancer, colorectal cancer, pancreatic cancer,melanoma, skin cancer, prostate cancer, cervical cancer, leukemia andbrain tumor.

Preferably, the tumor is non-small cell lung cancer. It is found by thepresent inventors that the polypeptide is specifically overexpressed ina tumor, whereas such the polypeptide is absent in a tumor-free tissue,thus a subject with a tumor specifically overexpressing the polypeptidecan be effectively diagnosed by the diagnostic system proposed inembodiments of the present disclosure. It is further discovered by thepresent inventors that the polypeptide is specifically overexpressed ina tumor selected from one or more of breast cancer, lung cancer,nasopharyngeal carcinoma, liver cancer, gastric cancer, esophagealcancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer,prostate cancer, cervical cancer, leukemia and brain tumor, thus theaccuracy of diagnosing the tumor as described above can be furtherimproved by using the diagnostic system proposed in the embodiment ofthe present disclosure. Meanwhile, it is demonstrated by the presentinventors that HLA-A0201 is present in Chinese populations in a highproportion, which binds with strong affinity to the polypeptide foractivation of a serious of immune responses, thus the accuracy ofdiagnosis of the polypeptide-expressing tumor from the HLA-A0201positive subject is improved by using the diagnostic system proposed inthe embodiment of the present disclosure.

The additional aspects and advantages of the present disclosure will bepartly described below, which will be apparent from the part descriptionor be understood by the practice of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the presentdisclosure will become apparent and readily understood from thedescription of examples of the present disclosure in combination withthe following drawings.

FIG. 1 is a schematic block diagram showing a diagnostic systemaccording to an embodiment of the present disclosure;

FIG. 2 is a graph showing affinity results of T2 cells loaded with thepolypeptide to HLA-A0201 detected by flow cytometry according to anembodiment of the present disclosure;

FIG. 3 is a graph showing activation of an immune response of CD8⁺ Tcell by the polypeptide verified by ELISPOTs method according to anembodiment of the present disclosure;

FIG. 4 is a graph showing specific killing of target cells loaded withthe polypeptide by activated CD8⁺ T cells according to an embodiment ofthe present disclosure; the sequence PLAFLIFNI is SEQ ID NO: 7; thesequence TLAFLIFNI is SEQ ID NO: 1; the sequence TMAFLIFNI is SEQ ID NO:2; the sequence TLAFLIFNV is SEQ ID NO: 3; the sequence TLAFLIFNL is SEQID NO: 4; the sequence TMAFLIFNL is SEQ ID NO: 5; and the sequenceTMAFLIFNV is SEQ ID NO: 6;

FIG. 5 is a graph showing the results of immunotherapy with thepolypeptide according to an embodiment of the present disclosure;

where panel A shows inhibition of tumor growth after treatment of anadjuvant alone group, an adjuvant+wild-type polypeptide (depicted inPLAFLIFNI, SEQ ID NO: 7) group, an adjuvant+mutant polypeptide (depictedin TLAFLIFNI, SEQ ID NO: 1), an adjuvant+mutant polypeptide's variant(depicted in TMAFLIFNI, SEQ ID NO: 2) group, an adjuvant+mutantpolypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3) group, anadjuvant+mutant polypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO:4) group, an adjuvant+mutant polypeptide's variant (depicted inTMAFLIFNL, SEQ ID NO: 5) group, and an adjuvant+mutant polypeptide'svariant (depicted in TMAFLIFNV, SEQ ID NO: 6) group, respectively,

panel B shows a mouse survival rate after treatment of an adjuvant alonegroup, an adjuvant+wild-type polypeptide (depicted in PLAFLIFNI, SEQ IDNO: 7) group, an adjuvant+mutant polypeptide (depicted in TLAFLIFNI, SEQID NO: 1), an adjuvant+mutant polypeptide's variant (depicted inTMAFLIFNI, SEQ ID NO: 2) group, an adjuvant+mutant polypeptide's variant(depicted in TLAFLIFNV, SEQ ID NO: 3) group, an adjuvant+mutantpolypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4) group, anadjuvant+mutant polypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO:5) group, and an adjuvant+mutant polypeptide's variant (depicted inTMAFLIFNV, SEQ ID NO: 6) group, respectively

FIG. 6 is a graph showing the results of immunotherapy with thepolypeptide according to an embodiment of the present disclosure,

where panel A shows inhibition of tumor growth after treatment ofdendritic cells (DCs) loaded with the wild-type polypeptide (depicted inPLAFLIFNI, SEQ ID NO: 7) group, DCs loaded with the mutation polypeptide(depicted in TLAFLIFNI, SEQ ID NO: 1) group, DCs loaded with the mutantpolypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO: 2) group, DCsloaded with the mutant polypeptide's variant (depicted in TLAFLIFNV, SEQID NO: 3) group, DCs loaded with the mutant polypeptide's variant(depicted in TLAFLIFNL, SEQ ID NO: 4) group, DCs loaded with the mutantpolypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5) group, andDCs loaded with the mutant polypeptide's variant (depicted in TMAFLIFNV,SEQ ID NO: 6) group, and

panel B shows a mouse survival rate after treatment of DCs loaded withthe wild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO: 7) group,DCs loaded with the mutation polypeptide (depicted in TLAFLIFNI, SEQ IDNO: 1) group, DCs loaded with the mutant polypeptide's variant (depictedin TMAFLIFNI, SEQ ID NO: 2) group, DCs loaded with the mutantpolypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3) group, DCsloaded with the mutant polypeptide's variant (depicted in TLAFLIFNL, SEQID NO: 4) group, DCs loaded with the mutant polypeptide's variant(depicted in TMAFLIFNL, SEQ ID NO: 5) group, and DCs loaded with themutant polypeptide's variant (depicted in TMAFLIFNV, SEQ ID NO: 6)group.

FIG. 7 is a graph showing the results of immunotherapy with thepolypeptide according to an embodiment of the present disclosure,

where panel A shows inhibition of tumor growth after immunotherapy withDCs infected with a lentiviral vector containing a nucleic acid sequenceencoding the wild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO:7), a lentiviral vector containing a nucleic acid sequence encoding themutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1), a lentiviralvector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO: 2), alentiviral vector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3), alentiviral vector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4), alentiviral vector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5) and alentiviral vector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TMAFLIFNV, SEQ ID NO: 6),respectively, and

panel B shows a mouse survival rate after immunotherapy with DCsinfected with a lentiviral vector containing a nucleic acid sequenceencoding the wild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO:7), a lentiviral vector containing a nucleic acid sequence encoding themutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1), a lentiviralvector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO: 2), alentiviral vector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3), alentiviral vector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4), alentiviral vector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5) and alentiviral vector containing a nucleic acid sequence encoding a mutantpolypeptide's variant (depicted in TMAFLIFNV, SEQ ID NO: 6),respectively.

FIG. 8 is a graph showing the results of immunotherapy with thepolypeptide according to an embodiment of the present disclosure,

where panel A shows inhibition of tumor growth after treatment of CTLsstimulated by the DCs loaded with the wild-type polypeptide (depicted inPLAFLIFNI SEQ ID NO: 7) group, CTLs stimulated by the DCs loaded withthe mutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1) group, CTLsstimulated by the DCs loaded with the mutant polypeptide's variant(depicted in TMAFLIFNI, SEQ ID NO: 2) group, CTLs stimulated by the DCsloaded with the mutant polypeptide's variant (depicted in TLAFLIFNV, SEQID NO: 3) group, CTLs stimulated by the DCs loaded with the mutantpolypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4) group, CTLsstimulated by the DCs loaded with the mutant polypeptide's variant(depicted in TMAFLIFNL, SEQ ID NO: 5) group and CTLs stimulated by theDCs loaded with the mutant polypeptide's variant (depicted in TMAFLIFNV,SEQ ID NO: 6) group as a vaccine, respectively; and

panel B shows a mouse survival rate after treatment of CTLs stimulatedby the DCs loaded with the wild-type polypeptide (depicted in PLAFLIFNISEQ ID NO: 7) group, CTLs stimulated by the DCs loaded with the mutantpolypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1) group, CTLs stimulatedby the DCs loaded with the mutant polypeptide's variant (depicted inTMAFLIFNI, SEQ ID NO: 2) group, CTLs stimulated by the DCs loaded withthe mutant polypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3)group, CTLs stimulated by the DCs loaded with the mutant polypeptide'svariant (depicted in TLAFLIFNL, SEQ ID NO: 4) group, CTLs stimulated bythe DCs loaded with the mutant polypeptide's variant (depicted inTMAFLIFNL, SEQ ID NO: 5) group and CTLs stimulated by the DCs loadedwith the mutant polypeptide's variant (depicted in TMAFLIFNV, SEQ ID NO:6) group as a vaccine, respectively.

DETAILED DESCRIPTION

The embodiments of the present disclosure are described in detail belowwith reference to the drawings, and the same or similar elements and theelements having identical or similar functions are denoted by likereference numerals throughout the descriptions. The embodimentsdescribed herein with reference to drawings are explanatory,illustrative, and used to generally understand the present disclosure.The embodiments shall not be construed to limit the present disclosure.

It should be noted that the terms “first” and “second” are used hereinfor purposes of description and are not intended to indicate or implyrelative importance or significance, impliedly indicate the quantity ofthe technical feature referred to. Thus, the features defined with“first” and “second” may explicitly or impliedly comprise one or moresuch features. In the description of the present disclosure, “aplurality of” means two or more than two this features, unless specifiedotherwise.

Polypeptide

In a first aspect of the present disclosure, provided in embodiments isan isolated polypeptide of SEQ ID NO: 1 (TLAFLIFNI) or a fragmentthereof. According to embodiments of the present disclosure, thefragment comprises at least 9 amino acids which has at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% sequence identity to the isolated polypeptide of SEQ ID NO: 1or has one or more of amino acid substitution(s), deletion(s) andaddition(s) compared to the isolated polypeptide of SEQ ID NO: 1.

The polypeptide proposed in embodiments of the present disclosure isderived from a mutant polypeptide in a tumor which is only present in atumor from a subject with mutation and is absent in a tumor-free tissue,thus exhibiting high specificity, and activating an immune response withhigh specificity. The CTLs produced in body by stimulation of thepolypeptide proposed in embodiments of the present disclosure kill tumorcells and tissues only, which do not affect normal tissues, therebyachieving accurate targeted therapy to tumor. Using the polypeptideproposed in embodiments of the present disclosure for tumorimmunotherapy achieves not only a good therapeutic effect, but also goodsafety and low side effect.

Particularly, according to an embodiment of the present disclosure, theone or more of amino acid substitution(s), deletion(s) and/oraddition(s) as described above is at least one of amino acidsubstitution at position 2 and amino acid substitution at position 9 inthe amino acid sequence as depicted in SEQ ID NO: 1. It is found by thepresent inventors that the amino acid substitutions at position 2 and/orposition 9 as depicted in SEQ ID NO: 1 does not change the specificitybetween the substituted amino acids and T cells, thus the immunogenicityof polypeptides is not changed.

More particularly, according to an embodiment of the present disclosure,the one or more of amino acid substitution(s), deletion(s) and/oraddition(s) as described above is at least one of substitution withMethionine at position 2 and substitution with Valine or Leucine atposition 9 in the amino acid sequence as depicted in SEQ ID NO: 1. Forexample, the fragment has 9 amino acids as depicted in any one of SEQ IDNO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ IDNO: 6.

According to an embodiment of the present disclosure, the polypeptidesdepicted in TLAFLIFNI (SEQ ID NO: 1), TMAFLIFNI (SEQ ID NO: 2),TLAFLIFNV (SEQ ID NO: 3), TLAFLIFNL (SEQ ID NO: 4), TMAFLIFNL (SEQ IDNO: 5) and TMAFLIFNV (SEQ ID NO: 6) each bind to HLA-A0201 with a highaffinity, thus having the ability to activate specific T cell immunity.It is further discovered by the present inventors that the variantsdepicted in TMAFLIFNI (SEQ ID NO: 2), TLAFLIFNV (SEQ ID NO: 3),TLAFLIFNL (SEQ ID NO: 4), TMAFLIFNL (SEQ ID NO: 5) and TMAFLIFNV (SEQ IDNO: 6), where the amino acid at position 2 is substituted withMethionine and/or the amino acid at position 9 is substituted withValine or Leucine, have an enhanced affinity with HLA-A0201, withoutaffecting the specificity between the polypeptides and T cells, and thusthe immunogenicity of polypeptides is not changed. Therefore, thepolypeptides depicted in both SEQ ID NO: 1 and SEQ ID NOs: 2-6 each havethe ability to activate specific T cell immunity.

Usage

In terms of application, in one aspect of the present disclosure,provided in embodiments is use of a reagent for detecting thepolypeptide as described above in the preparation of a kit fordiagnosing a tumor, use of the polypeptide as described above in thepreparation of a medicament for preventing or treating a tumor, or useof the polypeptide as described above in the preparation of a vaccinefor preventing or treating a tumor. Optionally, the polypeptide isexpressed in a tumor from a HLA-A0201 positive subject. Optionally, thetumor is one or more selected from the group consisting of breastcancer, lung cancer, nasopharyngeal carcinoma, liver cancer, gastriccancer, esophageal cancer, colorectal cancer, pancreatic cancer,melanoma, skin cancer, prostate cancer, cervical cancer, leukemia andbrain tumor. Preferably, the tumor is non-small cell lung cancer. Basedon the experiments and investigations, it is found by the presentinventors that the polypeptide as described above is specificallyoverexpressed in a tumor. Further, it is verified through experimentsand proposed by the present inventors that the kit prepared with thereagent for detecting the polypeptide as described above, or themedicament and the vaccine prepared with the polypeptide as describedabove, can be useful in diagnosing a tumor effectively, with highersafety and lower side effect. Meanwhile, it is surprisingly discoveredby the present inventors that the polypeptide binds with high affinityto HLA-A0201, and thus can be presented by HLA-A0201-expressingpresenting cells to CTL cells or TIL cells for activation of specific Tcell immunity, therefore when the polypeptide is expressed in a tumorfrom a HLA-A0201 positive subject, the safety and effectiveness ofdiagnosis by the kit or of treatment by the medicament or vaccine can besignificantly improved. Further, it is also discovered by the presentinventors that the polypeptide is specifically overexpressed in a tumorselected from one or more of lung cancer, melanoma, breast cancer,nasopharyngeal carcinoma, liver cancer, gastric cancer, esophagealcancer, colorectal cancer, pancreatic cancer, skin cancer, prostatecancer, cervical cancer, leukemia and brain tumor, especially non-smallcell lung cancer, thus when the tumor as described above is diagnosed ortreated, the effectiveness of diagnosis by the kit or of treatment bythe medicament or vaccine can be further improved.

In another aspect of the present disclosure, provided in embodiments isuse of the polypeptide as described above in the prevention or treatmentof a disease associated with a mutation of GLRA2 gene in a subject. Witha lot of screening experiments, it is found by the present inventorsthat the mutation occurring in the wild-type GLRA2 gene causes the aminoacid encoded at position 427 to be mutated into Threonine (i.e. Thr orT) from Proline (i.e. Pro or P). The polypeptides in embodiments of thepresent disclosure have same antigenic property as the polypeptideencoded by the mutated GLRA2 gene, which cause specific immune responseand allow the effector cells generated to specifically recognize andkill GLRA2 gene-mutated cells significantly, thus being capable ofpreventing or treating diseases associated with GLRA2 gene mutation.Also, it is demonstrated by the present inventors through experimentsthat the polypeptides in embodiments of the present disclosure canprevent or treat diseases associated with GLRA2 gene mutation, withsignificant efficacy.

Therapeutic Composition

In another aspect of the present disclosure, provided in embodiments isan isolated nucleic acid. According to an embodiment of the presentdisclosure, the isolated nucleic acid is a nucleic acid encoding thepolypeptide or the fragment thereof as described above, or a complementthereof, in which the isolated nucleic acid is capable of specificallyencoding the polypeptide as described above. As mentioned above, thepolypeptide binds with high affinity to HLA-A0201 and thus can bepresented by HLA-A0201-expressing presenting cells to CTL cells or TILcells for activation of specific T cell immunity. Therefore, thepolypeptide expressed by the isolated nucleic acid proposed in anembodiment of the present disclosure under a suitable condition can beused to prevent or treat a tumor, especially the polypeptide-expressingtumor from the HLA-A0201 positive subject, the safety and effectivenessof the treatment or prevention are improved.

Noted, it should be understood by skilled in the art that the nucleicacid mentioned in the description and claims of the present disclosurein fact refers to either or both chains of complementary double-strandsof the polypeptide. For convenience, only one chain is disclosed in mostcases in the description and claims of the present disclosure; howeverit means that another chain complemented is also disclosed. Besides, thenucleic acid in the present disclosure can be both in a DNA form and ina RNA form, and the disclosure to either form means both forms aredisclosed.

Accordingly, in another aspect of the present disclosure, provided inembodiments is a nucleic acid construct. According to an embodiment ofthe present disclosure, the nucleic acid construct includes an encodingsequence, in which the encoding sequence is the nucleic acid asdescribed above, and optionally a control sequence operably connected tothe encoding sequence, in which the control sequence is one or morecontrol sequences capable of directing the expression of the polypeptidein a host cell. According to an embodiment of the present disclosure,the control sequence includes but is not limited to U6 promoter, H1promoter, CMV promoter, EF-1 promoter, LTR promoter or RSV promoter.Therefore, after transfected or infected a suitable condition with thenucleic acid construct proposed in an embodiment of the presentdisclosure which is connected with a vector under, the suitable hostcell can efficiently express the polypeptide as described above, thusbeing capable of specifically preventing or treating a tumor in aneffective way, particularly the polypeptide-expressing tumor from theHLA-A0201 positive subject.

Accordingly, in another aspect of the present disclosure, provided inembodiments is an expression vector. According to an embodiment of thepresent disclosure, the expression vector includes the nucleic acidconstruct as described above, including but is not limited to aretrovirus vector, a lentiviral vector and/or an adenovirus-associatedvirus vector. Therefore, after transfected or infected with theexpression vector proposed in embodiments of the present disclosureunder a suitable condition, the host cell can efficiently express thepolypeptide as described above, thus being capable of specificallypreventing or treating a tumor in an effective way, particularly thepolypeptide-expressing tumor from the HLA-A0201 positive subject.

Accordingly, in another aspect of the present disclosure, provided inembodiments is a host cell. According to an embodiment of the presentdisclosure, the host cell carries the nucleic acid construct or theexpression vector as described above, optionally obtained bytransfecting or transforming the nucleic acid construct or theexpression vector, in which the transfection or transformation can beconducted by means of electrotransformation, viral transfection orcompetent cell transformation, depending on the properties of the hostcell and of the nucleic acid construct or the expression vector, as longas the polypeptide as described above can be efficiently expressed inthe host cell without affecting the good status of the host cell.According to an embodiment of the present disclosure, the host cell canefficiently express the polypeptide as described above under a suitablecondition, thus being capable of specifically preventing or treating atumor in an effective way, particularly the polypeptide-expressing tumorfrom the HLA-A0201 positive subject.

It should be noted, the term “suitable condition” described in thedescription of the present disclosure refer to the condition which issuitable for the expression of the polypeptide described in the presentdisclosure. It would be easily understood by skilled in the art that thecondition suitable for expressing the polypeptide includes but is notlimited to suitable method of transformation or transfection, suitableconditions for transformation or transfection, good status of host cell,appropriate density of the host cell, suitable environment andappropriate period for cell culture. The term “suitable condition” isnot particularly limited, and the condition for expressing thepolypeptide can be optimized by skilled in the art according to theparticular environment of the laboratory.

In still another aspect of the present disclosure, provided inembodiments is a pharmaceutical composition. According to an embodimentof the present disclosure, the pharmaceutical composition includes thepolypeptide as described above, and a pharmaceutically acceptableadjuvant. It is discovered by the present inventors through a lot ofexperiments that the pharmaceutical composition including thepolypeptide as described above and the pharmaceutically acceptableadjuvant can significantly stimulate the proliferation and secretion ofCTL cells or TIL cells, remarkably killing the tumor cell whichexpresses the polypeptide as an antigen, thus exhibiting an efficacy ofsignificantly preventing or treating a tumor, particularly the tumorspecifically overexpressing the polypeptide as an antigen.

In still another aspect of the present disclosure, provided inembodiments is an antigen-presenting cell. According to an embodiment ofthe present disclosure, the antigen-presenting cell can present thepolypeptide as described above. According to an embodiment of thepresent disclosure, the antigen-presenting cell presenting thepolypeptide can effectively induce an immune response by thetumor-specific polypeptide as described above (as an antigen) in asubject, thus activating the function of specific CTL killing. Theantigen-presenting cell proposed in an embodiment of the presentdisclosure has a remarkable efficacy of treating thepolypeptide-expressing tumor from the HLA-A0201 positive subject withsignificant therapeutic efficacy and high safety.

According to a particular embodiment of the present disclosure, theantigen-presenting cell is obtained by at least one of the followingsteps: contacting a cell capable of presenting an antigen with thepolypeptide, and introducing the nucleic acid, the nucleic acidconstruct or the expression vector as described above into a cellcapable of presenting an antigen. It is discovered by the presentinventors through experiments that the antigen-presenting cell canefficiently present the polypeptide by one or more means as describedabove, so that the polypeptide can be exposed on the surface of theantigen-presenting cell, thus being capable of efficiently inducing animmune response by the tumor-specific polypeptide as described above (asan antigen) in a subject, thereby further activating the function ofspecific CTL killing.

According to a particular embodiment of the present disclosure, theantigen-presenting cell is a dendritic cell which is characterized byhigh antigen endocytic and processing activity for presenting an antigenon the surface of cell. The dendritic cell is selected by the presentinventors as an antigen-presenting cell, which allows initiation,regulation and maintenance of a stronger immune response against thepolypeptide in the body.

In still another aspect of the present disclosure, provided inembodiments is an immune effector cell. According to an embodiment ofthe present disclosure, the immune effector cell can recognize thepolypeptide or the antigen-presenting cell presenting the polypeptide asdescribed above on its surface. According to an embodiment of thepresent disclosure, the immune effector cell is obtained by contactingthe antigen-presenting cell as described above with a cell withimmunogenicity. It is found by the present inventors that by such acontact, the antigen-presenting cell presenting the polypeptide iscapable of activating a non-activated cell with immunogenicity, thusgenerating a great amount of immune effector cells which specificallykills target cells expressing the polypeptide as an antigen. Accordingto another particular embodiment of the present disclosure, the cellwith immunogenicity is T lymphocyte, such as CD8⁺ T cell which can beactivated to a greater extend by the antigen-presenting cell, with theactivated CD8⁺ T cell having a stronger activity in specifically killingthe target cells expressing the polypeptide as an antigen.

In still another aspect of the present disclosure, provided inembodiments is a vaccine. According to an embodiment of the presentdisclosure, the vaccine includes the nucleic acid, the nucleic acidconstruct, the expression vector, the host cell, the antigen-presentingcell, or the immune effector cell as described above. As mentionedabove, after transfected or infected under a suitable condition with thenucleic acid, the nucleic acid construct or the expression vectorproposed in embodiments of the present disclosure, the host cell canexpress the polypeptide as described above, thus the nucleic acid, thenucleic acid construct, and the expression vector in embodiments of thepresent disclosure each can be used for preventing or treating thepolypeptide-expressing tumor; further the antigen-presenting cellproposed in embodiments of the present disclosure has a significantefficacy in treating the polypeptide-expressing tumor from the HLA-A0201positive subject; and furthermore the immune effector cell proposed inembodiments of the present disclosure has a significant efficacy inspecifically killing a target cell expressing the polypeptide as anantigen. Thus, the vaccine proposed in embodiments of the presentdisclosure which includes the nucleic acid, the nucleic acid construct,the expression vector, the host cell, the antigen-presenting cell or theimmune effector cell as described above, has a significant efficacy ofpreventing or treating the polypeptide-expressing tumor from theHLA-A0201 positive subject with higher safety and lower side effect.

In still another aspect of the present disclosure, provided inembodiments is an antibody. According to an embodiment of the presentdisclosure, the antibody specifically recognizes the polypeptide asdescribed above. The antibody proposed in an embodiment of the presentdisclosure can specifically bind to said polypeptide, thereby beingcapable of specifically identifying a tumor cell which specificallyoverexpresses the polypeptide, thus the antibody proposed in theembodiment of the present disclosure plays a huge role in tumordiagnosis, treatment or prevention. Further, according to an embodimentof the present disclosure, the antibody can be obtained by collectingserum of an animal immunized with the polypeptide as described above,and isolating the antibody of interest from the serum. With the methodfor preparing an antibody according to an embodiment of the presentdisclosure, the antibody specifically recognizing the polypeptide can beobtained effectively in a convenient and rapid way, which can be usedfor diagnosis, treatment or prevention of tumor effectively.

In summary, the polypeptide proposed in embodiments of the presentdisclosure has many advantages, for example, the polypeptide has higherspecificity against tumor due to exclusive presence in a tumor andabsence in a tumor-tissue sample of a subject, resulting in the higherspecificity of the immune response accordingly, with improved safety andlower side effect (i.e. rarely causing a severe adverse response)compared with other polypeptide vaccines against tumor; further it iseasily to be artificially synthesized due to its simple structure, thuscan be prepared as a vaccine or pharmaceutical composition againsttumor. The mutant polypeptide depicted in TLAFLIFNI (SEQ ID NO: 1) oreach of its variants can be used as a target or a vaccine forbiologically treating the polypeptide-expressing tumor from theHLA-A0201 positive subject, thus inducing an immune response in thebody. A composition of the polypeptide and an adjuvant, anantigen-presenting cell loaded with the polypeptide as a vaccine, or apolypeptide-specific DC-CTL or DC-CIK vaccine can be used tospecifically kill tumor cells, for preventing or treating a cancerexpressing the polypeptide, including but not limited to lung cancer,melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer, gastriccancer, esophageal cancer, colorectal cancer, pancreatic cancer, skincancer, prostate cancer, cervical cancer, leukemia, brain tumor and thelike.

Therapeutic Method

Further, provided in embodiments is a therapeutic method. According toembodiments of the present disclosure, the therapeutic method includesadministering to a subject a therapeutically effective amount of thepolypeptide, the nucleic acid, the nucleic acid construct, theexpression vector, the host cell, the pharmaceutical composition, theantigen-presenting cell, the immune effector cell, the vaccine or theantibody as described above. As mentioned above, the therapeutic methodproposed in an embodiment of the present disclosure includingadministering a therapeutically effective amount of any of thepolypeptide, the nucleic acid, the nucleic acid construct, theexpression vector, the host cell, the pharmaceutical composition, theantigen-presenting cell, the immune effector cell, the vaccine and theantibody as described above is capable of effectively treating orpreventing the polypeptide-expressing tumor from the HLA-A0201 positivesubject.

The terms “administer”, “administering”, “administered”,“administration” and the like as used herein refer to introducing apredetermined amount of a substance into a subject in any suitablemanner. The polypeptide, nucleic acid, nucleic acid construct,expression vector, host cell, pharmaceutical composition,antigen-presenting cell, immune effector cell, vaccine or antibody inembodiments of the present disclosure can be administered by any commonroute provided that they can reach the expected focus. Various routes ofadministration are contemplated, including but not limited toperitoneal, venous, muscular, subcutaneous, cortical, oral, topical,nasal, pulmonary and rectal. Further, the composition for oraladministration should be coated or formulated to prevent its activecomponent from degrading in the stomach. For example, the composition ofthe present disclosure can be administered in an injectable preparation.Furthermore, the pharmaceutical composition of the present disclosurecan be administered by using specific devices that deliver its activecomponent to target cells.

The frequency and dose of administration of the polypeptide, nucleicacid, nucleic acid construct, expression vector, host cell,pharmaceutical composition, antigen-presenting cell, vaccine or antibodyin embodiments of the present disclosure can be determined depending onthe type of disease to be treated, the route of administration, the age,sex, body weight of a subject, the severity of disease, and dosage formof medicament containing an active component. According to someembodiments of the present disclosure, the daily dose may be dividedinto 1 dose, 2 doses or multiple doses in a suitable form, foradministration once, twice or multiple times throughout the time period,provided a therapeutically effective amount is achieved.

The term “therapeutically effective amount” as used herein refers to anamount sufficient to significantly ameliorate certain symptomsassociated with a disease or condition, that is, an amount that providesa therapeutic effect for a given condition or dosage regimen. The terms“therapy”, “therapeutic”, “treatment”, “treat” and the like as usedherein refer to achieving a desired pharmacological and/or physiologicaleffect. As used herein, the “treatment” encompasses the administrationof the polypeptide, nucleic acid, nucleic acid construct, expressionvector, host cell, pharmaceutical composition, antigen-presenting cell,immune effector cell, vaccine or antibody in embodiments of the presentdisclosure to a subject for treatment, including but not limited toadminister the polypeptide, the nucleic acid, the nucleic acidconstruct, the expression vector, the host cell, the pharmaceuticalcomposition, the antigen-presenting cell, the immune effector cell, thevaccine or the antibody described in the present disclosure to anindividual in need thereof

Diagnostic Method

Further, provided in embodiments is a diagnostic method. According to anembodiment of the present disclosure, the diagnostic method includesdetecting whether a biological sample derived from a subject containsthe polypeptide or the fragment thereof as described above, anddetermining whether the subject suffers from a tumor based on thepresence or absence of the polypeptide in the biological sample. Thepolypeptide (as a tumor marker for cancer diagnosis due to its exclusivepresence in a tumor) free in serum can be detected by the massspectrometry, so as to determine whether a subject suffers from a canceror not. It is found by the present inventors that the polypeptide isspecifically overexpressed in a tumor, thus the diagnostic methodproposed in the embodiment of the present disclosure can be used toeffectively diagnose whether a subject suffers from a tumor where thepolypeptide is specifically overexpressed.

Furthermore, the present inventors still found that the polypeptide isspecifically overexpressed in a tumor selected from one or more of lungcancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer,gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer,skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor,especially non-small cell lung cancer, thus the accuracy of diagnosingthe tumor as described above can be further improved by using thediagnostic method proposed in the embodiment of the present disclosure.

Meanwhile, the present inventors yet still found that HLA-A0201 ispresent in Chinese populations in a high proportion, thus the accuracyof diagnosis of the polypeptide-expressing tumor from the HLA-A0201positive subject can be improved by using the diagnostic method proposedin the embodiment of the present disclosure.

Diagnostic System

Furthermore, provided in embodiments is a diagnostic system. Accordingto an embodiment of the present disclosure, referring to FIG. 1, thediagnostic system includes a polypeptide detecting device 100,configured to detect whether a biological sample derived from a subjectcontains the polypeptide as described above; and a diagnosis determiningdevice 200, connected to the polypeptide determining device 100 andconfigured to determine whether the subject suffers from a tumor basedon the presence or absence of the polypeptide in the biological sample.According to a particular embodiment of the present disclosure, whetherthe free polypeptide is present in serum of the subject can be detectedby a mass spectrometer, for determination of presence or absence of thefree polypeptide by an analysis device for mass spectrometry data, thusdetermining whether a subject suffers from a tumor. It is found by thepresent inventors that the polypeptide is specifically overexpressed ina tumor, thus the diagnostic system proposed in the embodiment of thepresent disclosure can be used to effectively diagnose whether a subjectsuffers from a tumor where the polypeptide is specificallyoverexpressed.

Furthermore, the present inventors still found that the polypeptide isspecifically overexpressed in a tumor selected from one or more of lungcancer, melanoma, breast cancer, nasopharyngeal carcinoma, liver cancer,gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer,skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor,especially non-small cell lung cancer, thus the accuracy of diagnosingthe tumor as described above can be further improved by using thediagnostic system proposed in the embodiment of the present disclosure.

Meanwhile, the present inventors yet still found that HLA-A0201 ispresent in Chinese populations in a high proportion, which binds withstrong affinity to the polypeptide for activation of a serious of immuneresponses, thus the accuracy of diagnosis of the polypeptide-expressingtumor from the HLA-A0201 positive subject is improved by using thediagnostic system proposed in the embodiment of the present disclosure.

It should be noted that the polypeptide according to embodiments of thepresent disclosure and the use thereof, the nucleic acid encoding thepolypeptide, nucleic acid construct, expression vector, host cell,pharmaceutical composition, antigen-presenting cell, immune effectorcell, vaccine and antibody, and the therapeutic method, diagnosticmethod and diagnostic system are discovered and achieved by the presentinventors through lots of creative labor and optimization.

The technical solutions of the present disclosure will be explainedbelow in combination with the embodiments. It will be appreciated byskilled in the art that the following examples are explanatory, andcannot be construed to limit the scope of the present disclosure. Theparticular techniques or conditions not specified in the examples can beperformed according to the techniques or conditions described inliteratures in the art or according to the product instructions. Inaddition, the reagents or instruments not indicated the manufacturer maybe commercially available, for example, obtained from Illumina.

EXAMPLE 1 Prediction of Polypeptide Affinity

With a lot of screening experiments, it is found by present inventorsthat the mutation occurring in the wild-type GLRA2 gene which encodes α2subunit of glycine receptor (i.e. a selective anion channel protein) ,causes the amino acid encoded at position 427 to be mutated intoThreonine (i.e. Thr or T) from Proline (i.e. Pro or P), where such theα2 subunit (distributed on cells of hippocampus, cerebral cortex,thalamus or other tissues) after bound with strychnine exhibitsextracellular ligand-gated ion channel activity and extracellularglycine-gated chloride channel activity. The mutated GLRA2 gene isspecifically expressed at a high level in tumor, and the mutantpolypeptide encoded is specific to tumor and is capable of binding toHLA-A0201 with high affinity. Such the binding activity has beenverified by the present inventors through experiments (shown as below).

In order to develop more valuable biologic drugs for clinic use,particular in cancer therapy, the present inventors selected a portionof the α2 subunit of glycine receptor including the amino acid Proline(i.e. Pro or P) at position 427 as the wild-type polypeptide (SEQ ID NO:7); selected a mutant polypeptide (SEQ ID NO: 1) where the amino acidProline (i.e. Pro or P) at position 427 is mutated into Threonine (i.e.Thr or T); and designed several variants based on the mutant polypeptide(SEQ ID NO: 1) with expectation of maintaining the high affinity toHLA-A0201 without sacrificing immunogenicity. Here only shows results ofthe wild-type polypeptides (SEQ ID NO: 7), the mutant polypeptide (SEQID NO: 1) and some mutant polypeptide' variants (SEQ ID NOs: 2-6) whichwere determined to be useful.

The affinity of the wild-type polypeptide (SEQ ID NO: 7), the mutantpolypeptide (SEQ ID NO: 1) or the mutant polypeptide' variants (SEQ IDNOs: 2-6) to a selected HLA allele (i.e., the subtype HLA-A0201) wasrespectively predicted by using independently developed software(prediction software of mutant polypeptide's affinity based on the tumorDNA and RNA sequencing, software copyright No: 2016SR002835), withprediction results represented in IC₅₀ scores, where IC₅₀ score below500 nM indicating presence of affinity, and IC₅₀ score below 50 nMindicating high affinity. The present inventors have predicted theaffinity of the wild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO:7), the mutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1), themutant polypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO: 2), themutant polypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3), themutant polypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4), themutant polypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5), andthe mutant polypeptide's variant (depicted in TMAFLIFNV, SEQ ID NO: 6),respectively, thus screening out those polypeptides whose IC₅₀ scoresare not only below 500 nM, but also lower than that of the wild-typepolypeptide. Table 1 shows the prediction results of affinity ofpolypeptides. Further, the affinity between the polypeptides screenedand T2 cells was verified in next steps.

TABLE 1 Prediction results of affinity betweenpolypeptides and HLA-A0201 Sequences of mutant Sequence of wild-typepolypeptide and its variants IC₅₀ (nM) polypeptide IC₅₀ (nM)TLAFLIFNI (SEQ ID NO: 1) 2.43 PLAFLIFNI (SEQ ID NO: 7) 12.10TMAFLIFNI (SEQ ID NO: 2) 2.89 PLAFLIFNI (SEQ ID NO: 7) 12.10TLAFLIFNV (SEQ ID NO: 3) 2.13 PLAFLIFNI (SEQ ID NO: 7) 12.10TLAFLIFNL (SEQ ID NO: 4) 2.56 PLAFLIFNI (SEQ ID NO: 7) 12.10TMAFLIFNL (SEQ ID NO: 5) 2.61 PLAFLIFNI (SEQ ID NO: 7) 12.10TMAFLIFNV (SEQ ID NO: 6) 2.99 PLAFLIFNI (SEQ ID NO: 7) 12.10

With prediction by the software, the mutant polypeptide and its variantsall have an IC₅₀ score below 50 nM with high affinity; further all theIC₅₀ scores are lower than that of the wild-type polypeptide (depictedin PLAFLIFNI, SEQ ID NO: 7), thus indicating that the affinity of thosepolypeptides is higher than that of the wild-type polypeptide.

EXAMPLE 2 Validation of Affinity between Polypeptide and T2 Cells

2.1 Synthesis and purification of polypeptides

The various types of polypeptides involved in the example of the presentdisclosure were synthesized by the standard solid phase peptidesynthesis, followed by purification with the reverse phase HPLC, whereits purity (>90%) is identified by the HPLC method and its identity isdetermined by the mass spectrometry.

2.2 Affinity Verification

T2 cells, as a hybridoma cell of HLA-A2 positive-T lymphocyte and Blymphocyte, can express HLA-A0201 on its surface, but could not transferendogenous antigens due to the deficiency of the essential transporterassociated with antigen processing (TAP) protein complex in theendogenous antigen presentation pathway. The T2 cells were purchasedfrom ATCC (Cat. No.: CRL-1992).

To a 24-well plate containing T2 cells (in 2×10⁵cells/well) resuspendedwith 500 μL serum-free Iscove's Modified Dulbecco's Medium (IMDM) whichcontains human β2 microglobulin in a final concentration of 3 μg/ml,added with the synthetic wild-type polypeptide (depicted in PLAFLIFNI,SEQ ID NO: 7), the mutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO:1), the mutant polypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO:2), the mutant polypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO:3), the mutant polypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO:4), the mutant polypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO:5), and the mutant polypeptide's variant (depicted in TMAFLIFNV, SEQ IDNO: 6) (each in a final concentration of 100 μM) respectively, followedby incubation in an incubator under 37° C. and 5% CO₂ overnight, induplicate for each group, where the well containing T2 cells alone isused as a background control, and the well containing T2 cells and CMVpolypeptide (depicted in NLVPMVATV, SEQ ID NO: 8) is used as a positivecontrol. After centrifugation at 200 g for 5 minutes for cellcollection, the collected cells were washed with phosphate buffer saline(PBS) buffer twice, before incubated with anti-HLA-A*02:01 FITCmonoclonal antibody at 4° C. for 30 minutes. The mean fluorescenceintensity of cells was then detected and analyzed by the flow cytometry(BD FACSJazz™)) and its software.

The fluorescence index (FI) was calculated using the following formula:

FI=[mean fluorescence intensity (MFI) of sample−MFI of background]/MFIof background,

where MFI of background represents the value in the absence ofpolypeptide, with FI>1.5 indicating high affinity between thepolypeptide and HLA-A*0201 molecule, 1.0 <FI<1.5 indicating moderateaffinity between the polypeptide and HLA-A*0201 molecule, and 0.5<FI<1.0 indicating low affinity between the polypeptide and HLA-A*0201molecule.

The detection results of affinity of the polypeptides are shown in Table2 and FIG. 2.

TABLE 2 Detection results of affinity between polypeptides and HLA-A0201Concentration Mean of fluorescence Samples polypeptide intensity FIConclusion PLAFLIFNI (SEQ ID NO: 7) 100 μM 650 1.63 high affinityTLAFLIFNI (SEQ ID NO: 1) 100 μM 643 1.60 high affinityTMAFLIFNI (SEQ ID NO: 2) 100 μM 660 1.67 high affinityTLAFLIFNV (SEQ ID NO: 3) 100 μM 620 1.51 high affinityTLAFLIFNL (SEQ ID NO: 4) 100 μM 615 1.49 moderate affinityTMAFLIFNL (SEQ ID NO: 5) 100 μM 690 1.79 high affinityTMAFLIFNV (SEQ ID NO: 6) 100 μM 677 1.74 high affinityBackground control   0 μM 247 0.00 no affinity CMV positive control100 μM 658 1.87 high affinity

It was verified by experiments that the FI of the background control andthe CMV positive control are respectively 0 and 1.87, both in a normalrange, while the FIs of the wild-type polypeptide (depicted inPLAFLIFNI, SEQ ID NO: 7), the mutant polypeptide (depicted in TLAFLIFNI,SEQ ID NO: 1), the mutant polypeptide's variant (depicted in TMAFLIFNI,SEQ ID NO: 2), the mutant polypeptide's variant (depicted in TLAFLIFNV,SEQ ID NO: 3), the mutant polypeptide's variant (depicted in TLAFLIFNL,SEQ ID NO: 4), the mutant polypeptide's variant (depicted in TMAFLIFNL,SEQ ID NO: 5), and the mutant polypeptide's variant (depicted inTMAFLIFNV, SEQ ID NO: 6) are each above 1.5, further demonstrating highaffinity of the wild-type polypeptide, as well as the mutant polypeptideand its five variants to HLA-A0201 molecule.

EXAMPLE 3 In Vitro Stimulation of CD8⁺ T Cells by Polypeptide

Peripheral blood mononuclear cells (PBMCs) in 100 mL peripheral bloodobtained from a HLA-A0201-positive healthy volunteer were isolated usingthe Ficoll solution, where CD8⁺ T cells among the PBMCs were isolatedwith CD8 magnetic beads, and monocytes among the PBMCs were incubated byan adherence method. Such the monocytes were induced into immaturedendritic cells (DCs) in the presence of GM-CSF (1000 U/ml) and IL-4(1000 U/ml), followed by inducing to polypeptide-specific mature DCs inthe presence of IFN-γ (100 U/ml) and CD40L (100 U/ml) as well as thesubsequently added with the mutant polypeptide (depicted in TLAFLIFNI,SEQ ID NO: 1), the mutant polypeptide's variant (depicted in TMAFLIFNI,SEQ ID NO: 2), the mutant polypeptide's variant (depicted in TLAFLIFNV,SEQ ID NO: 3), the mutant polypeptide's variant (depicted in TLAFLIFNL,SEQ ID NO: 4), the mutant polypeptide's variant (depicted in TMAFLIFNL,SEQ ID NO: 5), and the mutant polypeptide's variant (depicted inTMAFLIFNV, SEQ ID NO: 6) respectively.

The CD8⁺ T cells from the volunteer in a well were incubated with themature DCs obtained, along with IL-21, with addition of IL-2 and IL-7after 3 days' culture and addition IL-2 and IL-7 again at day 5 and day7 respectively, after which the collected mixture of cells were countedon day 10, for use in the next enzyme-linked immune-spot assay (ELISPOT)and lactate dehydrogenase (LDH) assay. The counting results are shown inTable 3.

TABLE 3 Results of cell counting after incubation Total Total numbernumber of cells of cells before after incubation incubationPLAFLIFNI (SEQ ID NO: 7) 2.0 × 10⁶ 1.01 × 10⁷ TLAFLIFNI (SEQ ID NO: 1)2.0 × 10⁶  9.6 × 10⁶ TMAFLIFNI (SEQ ID NO: 2) 2.0 × 10⁶  1.1 × 10⁷TLAFLIFNV (SEQ ID NO: 3) 2.0 × 10⁶ 1.18 × 10⁷ TLAFLIFNL (SEQ ID NO: 4)2.0 × 10⁶  1.2 × 10⁷ TMAFLIFNL (SEQ ID NO: 5) 2.0 × 10⁶  8.9 × 10⁶TMAFLIFNV (SEQ ID NO: 6) 2.0 × 10⁶  8.1 × 10⁶

With 10 days of incubation, the cells have proliferated significantly,about 4-6 times more than that before incubation.

EXAMPLE 4 ELISPOT Validation of CD8⁺ T Cell Immune Response Activated byPolypeptide

The mixture of cells collected in the example 3 were incubated with T2cells loaded with the mutant polypeptide (depicted in TLAFLIFNI, SEQ IDNO: 1) or the wild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO:7) respectively in an plate containing human Interferon-γ (IFN-γ) forthe ELISPOT assay, by which the spots produced were counted. The wellcontaining T2 cells loaded with the mutant polypeptide is anexperimental well, and the well containing T2 cells loaded with thewild-type polypeptide is a control well. The mutant polypeptide withimmunogenicity should meet the following requirement:

Spots number of mutant polypeptide/spots number of wild-type polypeptide>2,

-   -   where the number of spots produced by the mutant polypeptide        more than two times of the number of spots produced by the        wild-type polypeptide indicates the mutant polypeptide has        immunogenicity.

The detection results are shown in Table 4 and FIG. 3.

The reaction principle of ELISPOT assay is as follows: CD8⁺ T cells canspecifically recognize the complex of HLA-A0201 and the polypeptide,with different T cell populations recognizing different complexes ofHLA-A0201 and the polypeptides with different sequences, thus the CD8⁺ Tcell is capable of specifically recognizing the T2 cells loaded with themutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1), but could notrecognize the T2 cells loaded with the wild-type polypeptide (depictedin PLAFLIFNI, SEQ ID NO: 7). After specifically recognizing the complexof HLA-A0201 and the mutant polypeptide, the polypeptide specific-CD8⁺ Tcell is activated and secretes IFN-γ, which will be captured by thefirstly antibody coated on the ELISPOT plate and further be bound by anenzyme-coupled secondly antibody, and finally developed by addition of asubstrate which can be catalyzed by the enzyme, thus forming coloredspots. The number of spots produced represents the number of the IFN-γsecreted by the activated CD8⁺ T cell.

TABLE 4 Results of IFN-γ secreted by polypeptide-specific CD8+T cellunder the stimulation of polypeptide Number of spots produced Number ofRatio of Sequences of mutant by mutant spots mutant polypeptide orpolypeptide or produced by polypeptide its variants each of itswild-type to wild-type activating T cells variants polypeptidepolypeptide Conclusion TLAFLIFNI (SEQ ID NO: 1) 389 30 13 immunogenicTMAFLIFNI (SEQ ID NO: 2) 240 28 8.6 immunogenic TLAFLIFNV (SEQ ID NO: 3)267 34 7.9 immunogenic TLAFLIFNL (SEQ ID NO: 4) 373 40 9.3 immunogenicTMAFLIFNL (SEQ ID NO: 5) 347 42 8.2 immunogenic TMAFLIFNV (SEQ ID NO: 6)298 36 8.3 immunogenic

EXAMPLE 5 Demonstration of CD8⁺ T Cell Killing Activity by LDH ReleaseAssay

The mixture of cells collected in the example 3 were incubated with theT2 cells loaded with the mutant polypeptide (depicted in TLAFLIFNI, SEQID NO: 1), the mutant polypeptide's variant (depicted in TMAFLIFNI, SEQID NO: 2), the mutant polypeptide's variant (depicted in TLAFLIFNV, SEQID NO: 3), the mutant polypeptide's variant (depicted in TLAFLIFNL, SEQID NO: 4), the mutant polypeptide's variant (depicted in TMAFLIFNL, SEQID NO: 5), the mutant polypeptide's variant (depicted in TMAFLIFNV, SEQID NO: 6) and the wild-type polypeptide (depicted in PLAFLIFNI, SEQ IDNO: 7) respectively, or the T2 cells without polypeptide respectivelyfor 4 hours; after which 50 μL cell supernatant collected from each wellwas individually added into 50 μL substrate mixture of LDH forcatalyzation of the LDH substrate, and the absorbance at wavelengths of490 nm and 680 nm (as a reference) is measured, where a maximum releasewell, a volume correction well, a medium alone well, a spontaneousrelease well of effector cell and a spontaneous release well of targetwell as different control wells, as well effector cells (T cells) andtarget cells (T2 cell) in different ratios as experiment wells are setin triplicate.

The killing efficiency of CD8⁺ T cells to T2 cells is calculated by thefollowing formula:

Killing efficiency (%)=(experiment well−spontaneous release well ofeffector cell−spontaneous release well of target well+medium alonewell)/(maximum release well of target well−volume correctionwell−spontaneous release well of target well+medium alone well)×100%.

The reaction principle of the LDH release assay is as follows: thelactate dehydrogenase (LDH) is one of the endoenzyme in cytoplasm oflive cells, which could not penetrate the cell membrane under a normalcondition, however when the target cells are attacked and damaged by theeffector cells, resulting in the change of cell membrane permeability,the LDH can be released into the medium, thereby reducing the oxidizedcoenzyme I (NAD+) into NADH in the process of catalyzing lactic acid topyruvate, with the latter NADH formed into a colored formazan compound(which exhibits a high absorption peak at a wavelength of 490 nm or 570nm) in the presence of a hydrogen donor (phenazine dimethyl sulfate,PMS) and reduced Iodonitrotetrazolium chloride (INT) or NitrotetrazoliumBlue chloride (NBT), thus the viability of effector cells can becalculated according to the OD value. The results are shown in Table 5and FIG. 4.

TABLE 5 Results of specific recognition and killing oftarget cells Loaded with polypeptide by T cells Ratio of Ratio ofeffector effector cells to cells to target target Groups cells (1:1)cells (10:1) T cells (TLAFLIFNI, SEQ ID NO: 1) + T2 cells 3.13% 38.60%(TLAFLIFNI, SEQ ID NO: 1) T cells (TLAFLIFNI, SEQ ID NO: 1) + T2 cells2.73%  3.83% (PLAFLIFNI, SEQ ID NO: 7)T cells (TMAFLIFNI, SEQ ID NO: 2) + T2 cells 2.93% 35.06%(TMAFLIFNI, SEQ ID NO: 2) T cells (TMAFLIFNI, SEQ ID NO: 2) + T2 cells1.12%  4.51% (PLAFLIFNI, SEQ ID NO: 7)T cells (TLAFLIFNV, SEQ ID NO: 3) + T2 cells 3.97% 44.73%(TLAFLIFNV, SEQ ID NO: 3) T cells (TLAFLIFNV, SEQ ID NO: 3) + T2 cells4.26%  6.92% (PLAFLIFNI, SEQ ID NO: 7)T cells (TLAFLIFNL, SEQ ID NO: 4) + T2 cells 3.65% 47.13%(TLAFLIFNL, SEQ ID NO: 4) T cells (TLAFLIFNL, SEQ ID NO: 4) + T2 cell4.43%  5.56% (PLAFLIFNI, SEQ ID NO: 7)T cells (TMAFLIFNL, SEQ ID NO: 5) + T2 cells 2.90% 50.02%(TMAFLIFNL, SEQ ID NO: 5) T cells (TMAFLIFNL, SEQ ID NO: 5) + T2 cells1.90%  7.59% (PLAFLIFNI, SEQ ID NO: 7)T cells (TMAFLIFNV, SEQ ID NO: 6) + T2 cells 1.90% 30.59%(TMAFLIFNV, SEQ ID NO: 6) T cells (TMAFLIFNV, SEQ ID NO: 6) + T2 cells1.90%  4.56% (PLAFLIFNI, SEQ ID NO: 7)

As can be seen from Table 5 and FIG. 4, the T cells activated by themutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1), the mutantpolypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO: 2), the mutantpolypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3), the mutantpolypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4), the mutantpolypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5), or themutant polypeptide's variant (depicted in TMAFLIFNV, SEQ ID NO: 6) at aratio of effector cells to target cells of 1:1 or 10:1, is capable ofkilling the T2 cells loaded with the corresponding polypeptides, butcould not kill the T2 cells loaded with the wild-type polypeptide(depicted in PLAFLIFNI, SEQ ID NO: 7), thus further demonstrating the Tcells activated by the mutant polypeptide (depicted in TLAFLIFNI, SEQ IDNO: 1), the mutant polypeptide's variant (depicted in TMAFLIFNI, SEQ IDNO: 2), the mutant polypeptide's variant (depicted in TLAFLIFNV, SEQ IDNO: 3), the mutant polypeptide's variant (depicted in TLAFLIFNL, SEQ IDNO: 4), the mutant polypeptide's variant (depicted in TMAFLIFNL, SEQ IDNO: 5), and the mutant polypeptide's variant (depicted in TMAFLIFNV, SEQID NO: 6) are all capable of specifically killing the target cellsloaded with the corresponding polypeptide.

EXAMPLE 6 Establishment of Subcutaneously Implanted Tumor Model withH2087 Cell Line Expressing Polypeptide TLAFLIFNI (SEQ ID NO: 1) or eachof its Variants

6.1 Construction of a recombinant lentiviral vector expressingpolypeptide TLAFLIFNI (SEQ ID NO: 1) or each of its variants andpackaging of each corresponding lentivirus

The DNA sequence encoding the mutant polypeptide (depicted in TLAFLIFNI,SEQ ID NO: 1) is shown as TCGAGCTGCCTTCCCATTGGCCTTCCT (SEQ ID NO: 9);

the DNA sequence encoding a variant of the mutant polypeptide (depictedin TMAFLIFNI, SEQ ID NO: 2) is shown as TCGATGTGCCTTCCCATTGGCCTTCCT (SEQID NO: 10);

the DNA sequence encoding another variant of the mutant polypeptide(depicted in TLAFLIFNV, SEQ ID NO: 3) is shown asTCGAGCTGCCTTCCCATTGGCCTTGTA (SEQ ID NO: 11);

the DNA sequence encoding still another variant of the mutantpolypeptide (depicted in TLAFLIFNL, SEQ ID NO: 4) is shown asTCGAGCTGCCTTCCCATTGGCCTTCTA (SEQ ID NO: 12);

the DNA sequence encoding still another variant of the mutantpolypeptide (depicted in TMAFLIFNL, SEQ ID NO: 5) is shown asTCGATGTGCCTTCCCATTGGCCTTCTA (SEQ ID NO: 13);

the DNA sequence encoding still another variant of the mutantpolypeptide (depicted in TMAFLIFNV, SEQ ID NO: 6) is shown asTCGATGTGCCTTCCCATTGGCCTTGTA (SEQ ID NO: 14); and

-   -   the DNA sequence encoding the wild-type polypeptide (depicted in        PLAFLIFNI, SEQ ID NO: 7) is shown as CCAAGCTGCCTTCCCATTGGCCTTCCT        (SEQ ID NO: 15).

A lentiviral vector pHBLV-Puro expressing the wild-type polypeptide(depicted in PLAFLIFNI, SEQ ID NO: 7) was constructed and named aspHBLV-Puro-PLAFLIFNI;

a lentiviral vector pHBLV-Puro expressing the mutant polypeptide(depicted in TLAFLIFNI, SEQ ID NO: 1) was constructed and named aspHBLV-Puro-TLAFLIFNI;

a lentiviral vector pHBLV-Puro expressing a mutant polypeptide's variant(depicted in TMAFLIFNI, SEQ ID NO: 2) was constructed and named aspHBLV-Puro-TMAFLIFNI;

a lentiviral vector pHBLV-Puro expressing a mutant polypeptide's variant(depicted in TLAFLIFNV, SEQ ID NO: 3) was constructed and named aspHBLV-Puro-TLAFLIFNV;

a lentiviral vector pHBLV-Puro expressing a mutant polypeptide's variant(depicted in TLAFLIFNL, SEQ ID NO: 4) was constructed and named aspHBLV-Puro-TLAFLIFNL;

a lentiviral vector pHBLV-Puro expressing a mutant polypeptide's variant(depicted in TMAFLIFNL, SEQ ID NO: 5) was constructed and named aspHBLV-Puro-TMAFLIFNL; and

a lentiviral vector pHBLV-Puro expressing a mutant polypeptide's variant(depicted in TMAFLIFNV, SEQ ID NO: 6) was constructed and named aspHBLV-Puro-TMAFLIFNV.

In the presence of plasmids pSPAX2 and pMD2G, such seven lentiviralvectors each were used to co-transfect 293T cells for packaginglentivirus, thus obtaining lentivirus expressing the wild-typepolypeptide (depicted in PLAFLIFNI, SEQ ID NO: 7), lentivirus expressingthe mutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1), lentivirusexpressing the mutant polypeptide's variant (depicted in TMAFLIFNI, SEQID NO: 2), lentivirus expressing the mutant polypeptide's variant(depicted in TLAFLIFNV, SEQ ID NO: 3), lentivirus expressing the mutantpolypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4), lentivirusexpressing the mutant polypeptide's variant (depicted in TMAFLIFNL, SEQID NO: 5), and lentivirus expressing the mutant polypeptide's variant(depicted in TMAFLIFNV, SEQ ID NO: 6).

6.2 Establishment of human non-small cell lung cancer cell lineexpressing polypeptide TLAFLIFNI (SEQ ID NO: 1)

The human non-small cell lung cancer cell line H2087 which is HLA-A*0201positive was purchased from ATCC (Cat. No.: CRL 5922). The cell lineH2087 was incubated in the Dulbecco's Modified Eagle Medium (DMEM)containing 10% fetal calf serum, 100 U/mL penicillin and 100 U/mLstreptomycin in an incubator under 37° C. and 5% CO₂, and subsequentlyinfected with the lentivirus expressing the mutant polypeptide (depictedin TLAFLIFNI, SEQ ID NO: 1) as prepared in 6.1. Afterwards, the infectedcells were incubated with the Puromycin antibiotic for a certainduration to screen out those viable infected cells, i.e., obtaining thehuman non-small cell lung cancer cell line H2087 which expresses themutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1) and named asthe human non-small cell lung cancer cell line H2087 -polypeptideTLAFLIFNI (SEQ ID NO: 1).

6.3 Human immune reconstruction to NOD SCID mice

After isolated with Ficoll solution from 600 to 900 ml anticoagulantperipheral blood from a healthy volunteer, the collected PBMCs in aconcentration of 2×10⁷ cells/0.5 ml were intraperitoneally injected intoeach of 300 NOD SCID mice without immune leakage, so as to reconstruct ahuman immune system in the NOD SCID mice, with the mice after 4 weeksselected for subsequent inoculation of the human non-small cell lungcancer cell line H2087 -polypeptide TLAFLIFNI obtained in 6.2.

6.4 Construction of human non-small cell lung cancer-modeled mice

The human non-small cell lung cancer cell line H2087 -polypeptideTLAFLIFNI (SEQ ID NO: 1) obtained in 6.2 was incubated in DMEM mediumcontaining 10% fetal calf serum, 100 U/mL penicillin and 100 U/mLstreptomycin in an incubator under 37° C. and 5% CO₂, and then the cellswere centrifuged at 3000 rpm for collection. The collected cells werewashed with sterile physiological saline three times, and then dilutedappropriately. To 40 μl tumor cell suspension, 10 μl 0.4% phenol bluewas added for staining, subsequently the tumor cell suspension in aconcentration of 1×10⁸ cells/ml was prepared after counting undermicroscope. 100 μl of the tumor cell suspension was inoculated into eachof the NOD/SCID mice by subcutaneous injection at 4 weeks post immunereconstruction in 6.3, with daily observation of the presence/absence ofinfection at inoculation sites, the increase/decrease of tumor size (inthe case of presence of a tumor), and measurement of the major axis (a)and the minor axis (b) of a tumor by a vernier caliper every 2 or 3 days(where the tumor size=a×b×b/2). After 7 days from inoculation, a tumorin a size of rice grain was touchable at the inoculation site. Afterthat, the subcutaneous tumor-modeled NOD/SCID mice (modeled withH2087-polypeptide TLAFLIFNI (SEQ ID NO: 1)) were treated with apolypeptide+complete Freund's adjuvant vaccine, a polypeptide+DCsvaccine, a lentivirus-infected DCs vaccine and a DC-CTL vaccinerespectively, with the tumor size and mouse survival rate observed andrecorded every 2 days.

EXAMPLE 7 Preparation of Polypeptide Vaccine and Therapeutic Regimen

The subcutaneous tumor-modeled NOD/SCID mice obtained in 6.4 of theExample 6 were divided into 8 groups randomly as below, with 6 mice pergroup:

an adjuvant+wild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO: 7)group,

an adjuvant alone group,

an adjuvant+mutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1)group,

an adjuvant+mutant polypeptide's variant (depicted in TMAFLIFNI, SEQ IDNO: 2) group,

an adjuvant+mutant polypeptide's variant (depicted in TLAFLIFNV, SEQ IDNO: 3) group,

an adjuvant+mutant polypeptide's variant (depicted in TLAFLIFNL, SEQ IDNO: 4) group,

an adjuvant+mutant polypeptide's variant (depicted in TMAFLIFNL, SEQ IDNO: 5) group,

an adjuvant+mutant polypeptide's variant (depicted in TMAFLIFNV, SEQ IDNO: 6) group.

Except for the adjuvant alone group, mice in other 7 groups receivedfirst immunization, by subcutaneous injection at two sites of the mouseback with 100 μg wild-type polypeptide (depicted in PLAFLIFNI, SEQ IDNO: 7), 100 μg mutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1),100 μg mutant polypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO:2), 100 μg mutant polypeptide's variant (depicted in TLAFLIFNV, SEQ IDNO: 3), 100 μg mutant polypeptide's variant (depicted in TLAFLIFNL, SEQID NO: 4), 100 μg mutant polypeptide's variant (depicted in TMAFLIFNL,SEQ ID NO: 5), 100 μg mutant polypeptide's variant (depicted inTMAFLIFNV, SEQ ID NO: 6) respectively, each of which was mixed with 150p.1 Freund's complete adjuvant in PBS to a final volume of 300 pl. After2 weeks, differently-immunized mice in 7 groups received respectivesame-dosage booster immunizations at two week interval for 4 times intotal, with incomplete Freund's adjuvant for the latter threeimmunizations.

The general characteristics (including mental state, activity, response,diet, body weight and tumor growth) of the mice with tumor were observeddaily, where the major axis (a) and the minor axis (b) of a tumor weremeasured by a vernier caliper every 2 days for calculation of tumor sizeaccording to a formula of axbxb/2, where the mouse survival rate in atime period is calculated by the following formula:

Survival rate=the number of survival mice/(the number of survivalmice+the number of dead mice)×100%.

The results are shown in FIG. 5. It can be seen, the adjuvant+mutantpolypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1) group, theadjuvant+mutant polypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO:2) group, the adjuvant+mutant polypeptide's variant (depicted inTLAFLIFNV, SEQ ID NO: 3) group, the adjuvant+mutant polypeptide'svariant (depicted in TLAFLIFNL, SEQ ID NO: 4) group, the adjuvant+mutantpolypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5) group, andthe adjuvant+mutant polypeptide's variant (depicted in TMAFLIFNV, SEQ IDNO: 6) group are all capable of inhibiting tumor growth and prolongingmouse survival period effectively, compared to the adjuvant alone groupand the adjuvant+wild-type polypeptide (depicted in PLAFLIFNI, SEQ IDNO: 7) group.

EXAMPLE 8 Preparation of DC Polypeptide Vaccine and Therapeutic Regimen

Peripheral blood mononuclear cells (PBMCs) in 100 to 150 mLanticoagulant peripheral blood obtained from a healthy volunteer wereisolated with Ficoll solution. The collected PBMCs suspended in the RPMI1640 medium in a concentration of 2×10⁶-3×10⁶ cells/ml were incubated at37° C. for 2 hours, obtaining adherent monocytes which can be inducedinto DCs, and non-adherent peripheral blood lymphocytes (PBLs) for use.Such the monocytes were induced into immature DCs in the presence ofGM-CSF (1000 U/ml) and IL-4 (1000 U/ml), followed by inducing to matureDCs in the presence of IFN-γ (100 U/ml) and CD40L (100 U/ml), as well asthe subsequently added 10 μg/ml wild-type polypeptide (depicted inPLAFLIFNI, SEQ ID NO: 7), 10 μg/ml mutant polypeptide (depicted inTLAFLIFNI, SEQ ID NO: 1), 10 μg/ml mutant polypeptide's variant(depicted in TMAFLIFNI, SEQ ID NO: 2), 10 μg/ml mutant polypeptide'svariant (depicted in TLAFLIFNV, SEQ ID NO: 3), 10 μg/ml mutantpolypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4), 10 μg/mlmutant polypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5), 10μg/ml mutant polypeptide's variant (depicted in TMAFLIFNV, SEQ ID NO:6), respectively.

The subcutaneous tumor-modeled NOD/SCID mice obtained in 6.4 of theexample were randomly divided into seven groups as below, with 6 miceper group:

DCs loaded with the wild-type polypeptide (depicted in PLAFLIFNI, SEQ IDNO: 7) group,

DCs loaded with the mutant polypeptide (depicted in TLAFLIFNI, SEQ IDNO: 1) group,

DCs loaded with the mutant polypeptide's variant (depicted in TMAFLIFNI,SEQ ID NO: 2) group,

DCs loaded with the mutant polypeptide's variant (depicted in TLAFLIFNV,SEQ ID NO: 3) group,

DCs loaded with the mutant polypeptide's variant (depicted in TLAFLIFNL,SEQ ID NO: 4) group,

DCs loaded with the mutant polypeptide's variant (depicted in TMAFLIFNL,SEQ ID NO: 5) group,

DCs loaded with the mutant polypeptide's variant (depicted in TMAFLIFNV,SEQ ID NO: 6) group.

For each of the above seven groups, DCs (respectively loaded with thewild-type polypeptide, the mutant polypeptide or each of five mutantpolypeptide's variants) were washed with physiological saline threetimes and then diluted with the physiological saline to a concentrationof (4.0±0.5)×10⁷ cells/ml, and 0.1ml of the resulting diluent (in adosage of (4.0±0.5)×10⁶ cells) was administrated to each inner thighclose to the inguen of each mouse by intracutaneous injection, at oneweek interval for 2 injections in total. The vital signs of the micewere observed after administration, and the major axis (a) and the minoraxis (b) of a tumor were measured by a vernier caliper every 2 days forcalculation of tumor size according to a formula of a×b×b/2, with theweight change and survival rate observed and recorded.

The results are shown in FIG. 6. It can be seen, the DCs loaded with themutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO: 1) group, the DCsloaded with the mutant polypeptide's variant (depicted in TMAFLIFNI, SEQID NO: 2) group, the DCs loaded with the mutant polypeptide's variant(depicted in TLAFLIFNV, SEQ ID NO: 3) group, the DCs loaded with themutant polypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4)group, the DCs loaded with the mutant polypeptide's variant (depicted inTMAFLIFNL, SEQ ID NO: 5) group, and the DCs loaded with the mutantpolypeptide's variant (depicted in TMAFLIFNV, SEQ ID NO: 6) group areall capable of prolonging the mouse survival period and mitigating tumorgrowth significantly, compared to the DCs loaded with the wild-typepolypeptide (depicted in PLAFLIFNI, SEQ ID NO: 7) group.

EXAMPLE 9 Preparation of Lentivirus-Infected DCs Vaccine and TherapeuticRegimen

Peripheral blood mononuclear cells (PBMCs) in 100 to 150 mLanticoagulant peripheral blood obtained from a healthy volunteer wereisolated with Ficoll solution. After the collected PBMCs were incubatedat 37° C. for 2 hours and the non-adherent PBLs were removed, theadherent monocytes were cultured in the presence of recombinant humangranulocyte macrophage colony stimulating factor (rhGM-CSF) andrecombinant human interleukin—4 (rhIL-4). At Day 5, the medium waschanged with appropriate fresh medium where cell density was adjusted toa concentration of 1×10⁶ cells/ml, to which the lentivirus expressingthe wild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO: 7), thelentivirus expressing the mutant polypeptide (depicted in TLAFLIFNI, SEQID NO: 1), the lentiviral expressing a mutant polypeptide's variant(depicted in TMAFLIFNI, SEQ ID NO: 2), the lentiviral expressing amutant polypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3), thelentiviral expressing a mutant polypeptide's variant (depicted inTLAFLIFNL, SEQ ID NO: 4), the lentiviral expressing a mutantpolypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5) or thelentiviral expressing a mutant polypeptide's variant (depicted inTMAFLIFNV, SEQ ID NO: 6), respectively added. After 24 hours'incubation, the collected cells with lentivirus-containing mediumremoved were again incubated in a fresh medium containing 50 ng/mlrhIL-4, 100 ng/ml rh GM-C SF, 100U/ml IFN-γ and 100U /ml CD40L in anincubator under 37° C. and 5% CO₂ for 48 to 72 hours, so as to inducemature DCs, and then the DCs infected with lentivirus were observedunder the fluorescence microscope. The collected mature DCs were washedwith physiological saline three times and diluted with the physiologicalsaline to a concentration of (4.0±0.5)×10⁷ cells/ml for use. Thesubcutaneous tumor-modeled NOD/SCID mice obtained in 6.4 of the example6 were randomly divided into seven groups as below, with 6 mice pergroup:

DCs infected with the lentivirus expressing the wild-type polypeptide(depicted in PLAFLIFNI, SEQ ID NO: 7) group,

DCs infected with the lentivirus expressing the mutant polypeptide(depicted in TLAFLIFNI, SEQ ID NO: 1) group,

DCs infected with the lentivirus expressing a mutant polypeptide'svariant (depicted in TMAFLIFNI, SEQ ID NO: 2) group,

DCs infected with the lentiviral expressing a mutant polypeptide'svariant (depicted in TLAFLIFNV, SEQ ID NO: 3) group,

DCs infected with the lentiviral expressing a mutant polypeptide'svariant (depicted in TLAFLIFNL, SEQ ID NO: 4) group,

DCs infected with the lentiviral expressing a mutant polypeptide'svariant (depicted in TMAFLIFNL, SEQ ID NO: 5) group, and

DCs infected with the lentiviral expressing a mutant polypeptide'svariant (depicted in TMAFLIFNV, SEQ ID NO: 6) group.

For each of the above seven groups, the collected DCs (respectivelyinfected with the lentivirus expressing the wild-type polypeptide(depicted in PLAFLIFNI, SEQ ID NO: 7), the mutant polypeptide (depictedin TLAFLIFNI, SEQ ID NO: 1), the mutant polypeptide's variant (depictedin TMAFLIFNI, SEQ ID NO: 2), the mutant polypeptide's variant (depictedin TLAFLIFNV, SEQ ID NO: 3), the mutant polypeptide's variant (depictedin TLAFLIFNL, SEQ ID NO: 4), the mutant polypeptide's variant (depictedin TMAFLIFNL, SEQ ID NO: 5) or the mutant polypeptide's variant(depicted in TMAFLIFNV, SEQ ID NO: 6)) were washed with physiologicalsaline three times and then diluted with the physiological saline to aconcentration of (4.0±0.5)×10⁷ cells/ml, and 0.1ml of the resultingdiluent (in a dosage of (4.0±0.5)×10⁶ cells) was administrated to eachinner thighs close to the inguen of each mouse by intracutaneousinjection, at one week interval for 2 injections in total. The vitalsigns of the mice were observed after administration, and the major axis(a) and the minor axis (b) of a tumor were measured by a vernier caliperevery 2 days for calculation of tumor size according to a formula ofaxbxb/2, with the weight change and survival rate observed and recorded.

The results are shown in FIG. 7. It can be seen, the DCs infected withthe lentivirus expressing the mutant polypeptide (depicted in TLAFLIFNI,SEQ ID NO: 1) group, the DCs infected with the lentivirus expressing themutant polypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO: 2)group, the DCs infected with the lentiviral expressing the mutantpolypeptide's variant (depicted in TLAFLIFNV, SEQ ID NO: 3) group, theDCs infected with the lentiviral expressing the mutant polypeptide'svariant (depicted in TLAFLIFNL, SEQ ID NO: 4) group, the DCs infectedwith the lentiviral expressing the mutant polypeptide's variant(depicted in TMAFLIFNL, SEQ ID NO: 5) group and the DCs infected withthe lentiviral expressing the mutant polypeptide's variant (depicted inTMAFLIFNV, SEQ ID NO: 6) group are all capable of inhibiting tumorgrowth and prolonging mouse survival period effectively, compared to theDCs infected with the lentivirus expressing the wild-type polypeptide(depicted in PLAFLIFNI, SEQ ID NO: 7) group.

EXAMPLE 10 Preparation of Polypeptide-Specific DC-CTL Vaccine andTherapeutic Regimen

The CD8⁺ T cells sorted with CD8 magnetic beads from PBLs in the example8 were firstly stimulated by incubating with the DCs loaded with thewild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO: 7), the DCsloaded with the mutant polypeptide (depicted in TLAFLIFNI, SEQ ID NO:1), the DCs loaded with the mutant polypeptide's variant (depicted inTMAFLIFNI, SEQ ID NO: 2), the DCs loaded with the mutant polypeptide'svariant (depicted in TLAFLIFNV, SEQ ID NO: 3), the DCs loaded with themutant polypeptide's variant (depicted in TLAFLIFNL, SEQ ID NO: 4), theDCs loaded with the mutant polypeptide's variant (depicted in TMAFLIFNL,SEQ ID NO: 5) and the DCs loaded with the mutant polypeptide's variant(depicted in TMAFLIFNV, SEQ ID NO: 6), respectively, in a ratio of 1: 10of DCs: CD8⁺ T cell, in an incubator under 37° C. and 5% CO2 in thepresence of 500 IU/ml IL-2 and 50 ng/ml IL-7, with the second and thirdstimulations at week 2 and week 3 using DCs loaded with correspondingpolypeptides respectively and 500 IU/ml IL-2. The number of Tlymphocytes was counted at day 0, 7, 14 and 21 post incubation, forcalculation of cell proliferation index (PI), where PI=cell number afterproliferation/cell number seeded. The cytotoxic T lymphocytes (CTLs)were harvested at day 7 post the third stimulation, which were suspendedin 0.2 ml physiological saline, and then injected intravenously to thesubcutaneous tumor-modeled NOD/SCID mice via the tail in an amount of1×10⁸ cells/mouse. The vital signs of the mice were observed afteradministration, and the major axis (a) and the minor axis (b) of a tumorwere measured by a vernier caliper every 2 days for calculation of tumorsize.

The results are shown in FIG. 8. It can be seen, CTLs stimulated by theDCs loaded with the mutant polypeptide (depicted in TLAFLIFNI, SEQ IDNO: 1) group, CTLs stimulated by the DCs loaded with the mutantpolypeptide's variant (depicted in TMAFLIFNI, SEQ ID NO: 2) group, CTLsstimulated by the DCs loaded with the mutant polypeptide's variant(depicted in TLAFLIFNV, SEQ ID NO: 3) group, CTLs stimulated by the DCsloaded with the mutant polypeptide's variant (depicted in TLAFLIFNL, SEQID NO: 4) group, CTLs stimulated by the DCs loaded with the mutantpolypeptide's variant (depicted in TMAFLIFNL, SEQ ID NO: 5) group andCTLs stimulated by the DCs loaded with the mutant polypeptide's variant(depicted in TMAFLIFNV, SEQ ID NO: 6) group as a vaccine are all capableof inhibiting tumor growth and prolonging mouse survival periodsignificantly, compared to the CTLs stimulated by DCs loaded with thewild-type polypeptide (depicted in PLAFLIFNI, SEQ ID NO: 7) group.

INDUSTRIAL APPLICABILITY

The polypeptide of the present disclosure can be used in the preparationof a kit, a medicament or a vaccine, where such the medicament orvaccine prepared has higher specificity of the immune response comparedto other polypeptide vaccines against tumor, with improved safety andlower side effect (i.e. rarely causing a severe adverse response);further the polypeptide is easily to be artificially synthesized due toits simple structure, thus can be prepared as a vaccine orpharmaceutical composition against tumor.

Although embodiments of the present disclosure have been described indetail, it will be understood by those skilled in the art that variouschanges, modifications, substitutions and variations to the details canbe made in these embodiments in accordance with the teachings of thepresent disclosure, which are all within the scope of the presentdisclosure, and the scope of the disclosure is defined by the claims andtheir equivalents.

In the specification of the present disclosure, the terms “anembodiment”, “some embodiments”, “an example”, “a specific example”,“some examples” or “a particular embodiment” and the like are intendedto refer to particular features, structures, materials orcharacteristics described by way of example or embodiment are containedin at least one embodiment or example of the disclosure. In thisspecification, the schematic representation of the above terms does notnecessarily refer to the same embodiment or example. Moreover, theparticular features, structures, materials or characteristics describedmay be combined in any suitable manner in one or more of embodiments orexamples.

What is claimed is:
 1. A method of treating a tumor associated with a mutation in GLRA2 gene in an HLA-A0201 positive subject in need thereof, comprising: administering a therapeutically effective amount of a peptide or a nucleic acid encoding the peptide to the subject, wherein the peptide comprises the sequence of Thr-Leu-Ala-Phe-Leu-Ile-Phe-Asn-Ile (SEQ ID NO: 1) or a fragment thereof having at least 70% homology with SEQ ID NO:l.
 2. The method according to claim 1, wherein the fragment has one or two amino acid substitutions in the SEQ ID NO: 1 and the one or two amino acid substitutions are the substitution at one or both of position 2 and position 9 in the SEQ ID NO:
 1. 3. The method according to claim 1, wherein the peptide comprises a sequence having the structure: Thr-A-Ala-Phe-Leu-Ile-Phe-Asn-B, in which: A is Leu or Met, B is Ile, Val or Leu.
 4. The method according to claim 1, wherein the peptide is any one of: (SEQ ID NO: 1) TLAFLIFNI, (SEQ ID NO: 2) TMAFLIFNI, (SEQ ID NO: 3) TLAFLIFNV, (SEQ ID NO: 4) TLAFLIFNL, (SEQ ID NO: 5) TMAFLIFNL, and (SEQ ID NO: 6) TMAFLIFNV.


5. The method according to claim 1, wherein the nucleic acid encoding the peptide is any one of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO:
 14. 6. The method according to claim 1, wherein a composition comprising the peptide and a pharmaceutically acceptable adjuvant is administered to the subject in response to the administration of the peptide.
 7. The method according to claim 1, wherein dendritic cells (DCs) that have been incubated with the peptide are administered.
 8. The method according to claim 1, wherein an expression vector comprising the nucleic acid encoding the peptide is administered to the subject in response to the administration of the nucleic acid encoding the peptide.
 9. The method according to claim 8, wherein the expression vector includes a retrovirus vector, a lentiviral vector or an adenovirus-associated virus vector.
 10. The method according to claim 1, wherein the tumor is selected from breast cancer, lung cancer, nasopharyngeal carcinoma, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor. 